Development of novel compounds for the molecular imaging of hypoxia: 18F-labeled nitroimidazoles and hypoxia inducible factor 1 alpha (HIF-1a)-targeting compounds

Abstract

Imaging of hypoxia has been previously achieved using positron emission tomography (PET) with radiotracers such as [18F]fluoroazomycin arabinoside ([18F]FAZA) [1] or [18F]fluoromisonidazole ([18F]FMISO) [2]. These typical nitroimidazole containing compounds are reductively trapped in hypoxic areas. Herein, we propose two novel ways to imaging hypoxia, one with a novel 2-nitroimidazole of N-(4-[18F]fluorobenzyl)-2-(2-nitro-1H-imidazol-1-yl)-acetamide ([18F]FBNA). Secondly, we propose to directly image the master regulator of hypoxia of hypoxia inducible factor 1 alpha (HIF-1a). We will comparatively assess the imaging of HIF-1a versus traditional imaging agents of [18F]FMISO, [18F]FAZA and the novel 2-nitroimidazole imaging agent of [18F]FBNA. We have developed two lead structures that bind to HIF-1a, and have radiolabelled them using [18F]fluorine prosthetic group chemistry. [18F]SFB-link-c-(Ppg)LLFVY was prepared using the prosthetic group of [18F]SFB and the primary amine containing peptide link-c-(Ppg)LLFVY. [18F]SFB-link-c-(Ppg)LLFVY was prepared in 66.2 ± 5.4% decay corrected recovered radiochemical yield from [18F]fluoride (n=8) with a purity >99%. [18F]FPeP4 was prepared using Pd-mediated SONOGASHIRA cross-coupling reaction between [18F]FPA and N-(4-iodophenyl)-4-((4-(pyridine-3-ylmethyl)piperazin-1-yl)methyl)benzamide. [18F]FPeP4 was prepared in 5 ± 2% decay corrected recovered radiochemical yield from [18F]fluoride (n=5) with a purity >99%. Novel nitroimidazole [18F]FBNA was synthesized starting from the prosthetic group of [18F]FBAmine. [18F]FBNA was prepared in 47.4 ± 5.4% decay corrected recovered radiochemical yield from [18F]FBAmine (n=8) with a purity >99%. Radiopharmacological profile of [18F]FPeP4, [18F]SFB-link-c-(Ppg)LLFVY and [18F]FBNA were evaluated in comparison to [18F]FAZA and [18F]FMISO using triple negative breast cancer cell line MDA-MB-231. In HIF-1a overexpressing MDA-MB-231 cells, cellular uptake of [18F]SFB-link-c-(Ppg)LLFVY reached an internalized maximum of 30% applied radioactivity/mg protein at 120 min and [18F]FPeP4 reached an internalized maximum of 400% applied radioactivity/mg protein at 120 min. In non-transfected hypoxic MDA-MB-231 cells, [18F]FBNA reached an internalized maximum of 9% applied radioactivity/mg protein at 120 min in hypoxic conditions. Radiopharmacological evaluation of all compounds included small animal PET imaging. Small animal PET studies of both HIF-1a targetting radiotracers demonstrated poor tumor uptake compared to [18F]FAZA, [18F]FMISO and [18F]FBNA. This was due to the poor pharmacokinetics of these radiotracers in vivo, with both radiotracers exhibiting large amounts of first pass liver trapping. [18F]FPeP4 demonstrated a SUVmax of 0.3 in MCF7 tumor bearing NIH-III mice compared to [18F]FBNA which had a SUVmax of 0.75. Tumor to muscle ratio of [18F]FBNA in MFC-7 tumor model was 3 which was equal to [18F]FPeP4 at 3. Yet, [18F]FBNA was found to be metabolically unstable, whereas [18F]FPeP4 was metabolically stable. [18F]SFB-link-c-(Ppg)LLFVY and [18F]FPeP4 are the first novel radiotracers to be designed that bind to HIF-1a. Their in vitro uptake showed increased uptake in HIF-1a overexpressing cells, yet their biodistribution in vivo is currently poor. Improvements to the delivery of these radiotracers in vivo could improve their future usage as radiotracers targeting HIF-1a.